突变型与野生型HPV16 DNA疫苗的免疫原性

目的评价突变人乳头瘤病毒16(HPV16)E7锌指结合区后HPV16E7 DNA疫苗的免疫原性.方法将构建的野生型(pcDNA3.1/16E7)和突变型(pcDNA3.1/16ME7)DNA疫苗经肌肉免疫C57BL/6小鼠,分离脾单个核细胞,用E7特异性多肽刺激后,测定E7特异性白介素-2(IL-2)和干扰素-γ(IFN-γ)的水平及细胞毒性T淋巴细胞(CTL)反应.同时以未加E7多肽组作对照组.结果经E7特异性多肽刺激后,pcDNA3.1/16ME7产生IL-2的斑点数明显高于pcDNA3.1/16E7、pcDNA3.1和空白对照组.pcDNA3.1/16ME7的斑点数是pcDNA3.1/16E7的5倍多,两组间差异有显著性(P＜0.05);pcDNA3.1/16ME7产生的IFN-γ是pcDNA3.1/16E7的2倍,差异有显著性(P＜0.05);LDH结果显示在E:T为45:1时,pcDNA16/E7、pcDNA16/ME7、pcDNA3.1及未经免疫的小鼠4组之间CTL细胞杀伤率分别为(28.7±1.2)%、(55±2.2)%、(12.5 2.0)%和(11.5±1.2)%,前两组与后两组中任一组之间差异均有显著性,前2组之间差异亦有显著性(P＜0.05).而未加E7多肽组,各组间差异均无显著性(P＞0.05).结论突变HPV16E7锌指结合区能显著增强DNA疫苗的免疫原性,是提高DNA疫苗免疫原性的策略之一.     

人乳头瘤病毒16型E7DNA疫苗的构建及其诱导特异性淋巴细胞增殖的研究

目的 :探讨人乳头瘤病毒 16型 (HPV16 )E7DNA疫苗诱导机体特异性细胞免疫应答的情况。方法 :采用分子克隆技术 ,构建HPV16野生型E7基因的真核表达重组体 ,将其转化大肠杆菌JM10 9进行筛选 ,通过限制性内切酶酶切鉴定和PCR分析 ,证明重组质粒中目的基因插入片段及载体DNA大小、方向、插入位点均正确 ,获得HPV16E7DNA疫苗 ,将E7DNA疫苗经皮内注射免疫动物 ,MTT比色法体外检测特异性淋巴细胞增殖反应。结果 :野生型E7DNA疫苗免疫组脾淋巴细胞在体外受到E7蛋白的再次刺激后出现特异性淋巴细胞增殖反应。结论 :野生型E7DNA疫苗可诱导特异性的细胞免疫应答 ,皮内注射HPVDNA疫苗是一种简便有效的免疫接种途径     

丙型肝炎病毒多CTL表位DNA疫苗免疫学特性的初步研究

目的:探讨丙型肝炎病毒(HCV)疫苗研制并为抗HCV感染提供实验依据。方法:从HCV J株基因组中,选取小鼠(H-2d)CTL表位基因序列,以pcDNA3.1(+)质粒为载体,构建相应的真核表达质粒,经肌肉免疫后,取受免疫小鼠血清和脾细胞,用ELISA及MTT法检测小鼠血清中IL-2和IFN水平及小鼠脾细胞的增殖活性。结果:实验组小鼠血清中IL-2及IFN水平明显高于对照组[IL-2:(208±8.88)比(106±6.06);IFN:(179.5±2.52)比(90.5±2.52),P<0.05];实验组淋巴细胞增殖指数明显高于对照组(1.56比1.13,P<0.05)。结论:本实验构建的pcDNA3.1(+)/HCV CTL真核表达质粒,经肌肉免疫后可以使受免疫小鼠细胞免疫应答水平提高,淋巴细胞增殖活性增高。     

IL-12对乙肝病毒全S基因疫苗免疫效应的影响

目的：探讨IL-12对乙型肝炎病毒全S基因疫苗的免疫效果的影响。 方法：BABL/c小鼠分为对照组、HBV全S基因表达质粒单独免疫组（pcDNA3-PS）及pcDNA3-PS联合IL-12表达质粒免疫组（pcDNA3-PS＋IL-12）,免疫后6周测血清抗HBs,T细胞增殖反应实验及IL-2和IFN-γ水平。 结果：pcDNA3-PS＋IL-12组抗HBs滴度、刺激指数（SI）、细胞因子水平均较pcDNA3-PS增高,差异有显著性;两免疫组抗HBs滴度、SI和细胞因子水平均较对照组增高,差异有显著性。 结论：HBV全S基因疫苗可刺激小鼠产生特异性免疫,IL-12对HBV全S基因疫苗具有免疫佐剂效果。     

融合DNA疫苗CRT180/HPV6E7的构建及其免疫学特性研究

目的 构建融合DNA疫苗CRT180/HPV6E7,并评价其体外实验和动物模型上的免疫学特性,尤其是抗病毒致病基因的特异性细胞免疫反应.方法 分别构建DNA重组体pcDNA3.1-CRT180/HPV6E7,pcDNA3.1-CRT180,pcDNA3.1-HPV6E7.将pcDNA3.1-HPV6E7质粒经脂质体介导转染B16细胞,以G418筛选阳性细胞集落,荧光显微镜观察和RT-PCR鉴定表达HPV6E7的阳性细胞株.将质粒DNA肌注免疫C57BL/6小鼠,3次后取全血经流式细胞仪检测T细胞亚群的变化,LDH法检测小鼠的脾细胞和淋巴细胞与靶细胞B16/HPV6E7孵育后的CTL活性以及ELISA法检测共孵育上清中IL-2和IFN-γ浓度的变化.结果 各组质粒经鉴定表明构建正确.转染后细胞株稳定表达HPV6E7.DNA疫苗免疫小鼠后,pcDNA3.1-CRT180/HPV6E7免疫组较pcDNA3.1-HPV6E7免疫组的外周血CD8 T细胞和TCRγδT细胞的比例显著增加,脾细胞和淋巴细胞针对靶细胞B16/HPV6E7的CTL杀伤活性更明显,分泌IL-2和IFN-γ的水平升高(P＜0.05).结论 CRT180与HPV致病基因6E7相连的重组质粒疫苗pcDNA3.1-CRT180/HPV6E7能引发小鼠T细胞亚群CD8 分化并诱导出显著的特异性CTL反应.推测CRT180/HPV6E7 DNA疫苗在动物模型上可以有效激活抗HPV特异性细胞免疫,有利于病毒感染的清除.     

CD40L融合改造后的人乳头瘤病毒16型E7基因DNA疫苗的构建及其免疫原性测定

目的研究针对人乳头瘤病毒(HPV)16型的DNA疫苗,测定其免疫原性,用于治疗HPV16感染及感染相关恶性肿瘤。方法联合采用基因切割重排、定点突变及密码子优化等策略改造HPV16的转化基因E7,基因合成法获得改造后的E7基因(mE7);PCR法将mE7基因与CD40L胞外区编码序列融合,然后以pVR1012为载体构建pVR1012-mE7(mE7)及pVR1012-mE7/CD40L(mE7/CD40L)表达质粒;经肌肉免疫C57BL/6小鼠;ELISA法检测E7特异性血清抗体水平,ELISPOT法分析E749-57(H-2b)特异性分泌IFN-γ的CD8 T细胞活化水平,胞内染色-流式细胞检测分析E7特异性CD4 Th细胞活化水平;并在C57BL/6小鼠体内进行疫苗抗瘤活性检测。结果与野生型E7基因(wE7)相比,mE7基因诱发产生的E7特异抗体水平(P<0.01)、分泌IFN-γ的CD8 T细胞数目(P<0.01)及CD4 Th细胞活化水平(P<0.05)均显著提高;与mE7基因相比,mE7/CD40L融合基因可进一步显著提高E7特异性分泌IFN-γ的CD8 T细胞数目(P<0.01),但对E7特异性抗体产生及CD4 Th细胞活化水平没有明显影响。疫苗小鼠体内预防性免疫实验中,经wE7免疫的小鼠接种瘤细胞后2周内全部形成移植瘤,而所有经mE7及mE7/CD40L免疫的小鼠在瘤细胞攻击后第7周仍未见移植瘤形成;疫苗体内治疗性免疫实验中,小鼠在接种wE7后第8天左右全部形成移植瘤,并呈渐进性生长,而接种mE7的小鼠移植瘤清除率为30%,接种mE7/CD40L的小鼠移植瘤清除率增高至45%。对移植瘤的组织学检查结果显示,mE7/CD40L及mE7免疫组小鼠瘤细胞间及瘤组织周围可见大量淋巴细胞浸润,而wE7组小鼠的瘤细胞呈编织状紧密排列,未见有淋巴细胞浸润。结论HPV16型mE7/CD40L融合基因疫苗免疫小鼠后可诱发较强的E7特异性细胞免疫及体内抗瘤活性,具有较强的免疫原性。     

人乳头瘤病毒16型E2蛋白真核载体的构建与表达

目的构建人乳头瘤病毒16型（HPV16）E2蛋白真核载体,为研究其基因疫苗免疫活性奠定实验基础。方法 PCR扩增HPV16 E2基因片段,将其连接到真核表达载体pcDNA3.1（＋）,双酶切及测序鉴定。将质粒pcDNA3.1（＋）与pcDNA3.1（＋）/HPV16 E2分别转染HeLa细胞,RT-PCR鉴定E2基因在HeLa细胞中的表达。结果 HPV16 E2基因片段插入到pcDNA3.1（＋）相同酶切位点,即构建了真核表达载体pcDNA3.1（＋）/HPV16 E2;转染pcDNA3.1（＋）/HPV16 E2的细胞,检测到HPV16 E2 mRNA。结论构建的真核表达载体pcDNA3.1（＋）/HPV16 E2能在HeLa细胞内有效表达。     

人类免疫缺陷病毒DNA疫苗黏膜免疫小鼠免疫力研究

目的以人类免疫缺陷病毒1型(HIV-1)疫苗免疫BALB/c(H-2d)小鼠为模型,探讨有效活化黏膜与系统免疫力的策略。方法将6~8周龄BALB/c小鼠随机分为6组,每组3只;利用DNA疫苗加或不加疫苗佐剂滴鼻途径进行黏膜引导免疫,利用重组痘苗天坛株疫苗进行系统加强的策略以提高机体免疫反应。DNA疫苗免疫时间是0、14、28d,剂量每只小鼠10μg/次;重组痘苗病毒加强免疫时间是第42天,剂量每只小鼠1×107PFU/次。初次免疫后第56天处死小鼠。ELISPOT和胞内染色测定T淋巴细胞免疫反应;ELISA法测血清特异的IgG和鼻咽部、肺部灌洗液特异的IgA。结果单独DNA疫苗黏膜免疫后只活化微弱的系统T细胞免疫(2±1)斑点形成细胞(spotformingcells,SFC/106个细胞),黏膜共同施用霍乱毒素可提高相应的系统T细胞免疫力(14±11SFC/106个细胞),重组痘苗天坛株疫苗系统加强后,分泌γ-干扰素(IFN-γ)的特异性T淋巴细胞升高4·5倍(61±35SFC/106个细胞);胞内染色可获得1·8%±1·4%HIV-1Gag特异性CD8+T细胞免疫反应,同时,血清中特异性IgG抗体与肺部灌洗液的特异性IgA抗体水平在痘苗天坛株疫苗系统加强后分别提高2倍吸光度(A)值:1·5±0·3和2·5倍(A值:1·8±0·8)。结论黏膜进行引导免疫并辅以系统加强的免疫策略可诱发有效的黏膜、系统体液免疫反应和T淋巴细胞免疫。     

Immune responses to the expressed products of the CSP antigen gene of Plasmondium falciparum south in China isolate FCC1/HN in Hela cell

Immune responses to the expressed products of the CSP antigen gene of Plasmondium falciparum south in China isolate FCC1/HN in Hela cell@Yu XB @Liu YW @Luo SH     

人乳头瘤病毒6b型病毒样颗粒免疫活性的研究

目的：了解人乳头瘤病毒（HPV）6b型病毒样颗粒（VLP）的免疫活性及其诱导的保护性抗体对HPV11型VLP和牛乳头瘤病毒1型（BPV1）VLP的交叉免疫反应。方法：将HPV6b、HPV11和BPV1晚期基因L1的开放读码框架（ORFs）分别重组到杆状病毒中,该重组病毒在感染的昆虫细胞（Sf9细胞）中表达,表达的L1蛋白可自行组装成HPV6b、HPV11和BPV1 VLP。取50μg纯化的HPV6bVLP分别在第0天和第21天经肌肉免疫Balb/c鼠。第二次免疫后1周及3个月取血,检测血清抗HPV6b、HPV11和BVP1 VLP和BPV1 VLP IgG滴度;血凝集抑制试验检测HPV6bVLP免疫产生的抗血清是否能阻止HPV11 VLP和BPV1 VLP与鼠红细胞凝集。结果：免疫后1周,应用ELISA方法检测抗HPV6b VLP、HPV11 VLP和BVP1 VLP血清IgG滴度分别为1：6400、1：600和1：600。3个月后,抗HPV6bVLP、HPV11 VLP 和BVP 1VLP血清IgG滴度分别为1：800、1：400和1：100。血凝集抑制试验显示HPV6bVLP产生的抗血清能够阻止高度同源性的HPV6b VLP和HPV11 VLP与鼠红细胞结合。结论：HPV6b VLP具有很强的免疫原性,免疫后产生的抗血清具有阻止HPV6b VLP和HPV11 VLP与细胞结合的能力。HPV6b和11型间确实存在交叉反应的中和表位,HPV6b VLP有可能用于防治HPV6b及HPV11感染的疫苗。     

CONSTRUCTION AND IMMUNOGENICITY OF HUMAN PAPILLOMAVIRUS TYPE 6B L1 RECOMBINANT PLASMID

Objective To construct a DNA vaccine as a prophylactic model to prevent condyloma acuminatum and detect its immunogenicity in mice. Methods The major capsid protein (L1) gene of human papillomavirus (HPV) 6b was inserted into an eukaryotic expression plasmid (pcDNA3.1). The recombinant plasmid was transfected into COS-7 cells. Western blot were performed to detect whether L1 protein can be expressed in eukaryotic cells. Eighteen female BALB/c mice were tested for immunogenicity study. Results The recombinant plasmid (pcDNA3. 1-HPV6bL1) was verified as HPV6b L1 gene by sequencing. Western blot showed specific strip. Anti-L1 protein antibodies could be detected in the mice‘s sera inoculated with pcDNA3.1-HPV6bL1. Similarly, IL-4, IL-2, and IFN-γ were increased in the same mice. Conclusion HPV6b L1 recombinant plasmid was constructed successfully which had immunogenicity for BALB/c mice. It provided experimental evidence for the research of DNA vaccine of condyloma acuminata.     

Immune response induced by HPV18 E2 DNA vaccine combined with IL-12

Objective:To study the effectiveness of human papillomavirus type 18 E2 gene (HPV18-E2) vaccine and interleukin-12(IL-12) on their immune effects. Methods:Thirty-five Balb/c female mice aged 6-8-week-old were randomly divided into five groups (each n =7).The mice were given instramuscular injection of DNA vaccines at 2-week four 4 times(100mg/time).The tails were cut to draw blood at the 0,2,4,6 weeks,and then the mice were executed by eyeball blood letting at 8 weeks.ELISA was used for the quantitative detection of the specific lgG antibody in the srea of Balb/c mice and the cytokine IFN-γ and IL-4 in mice MTT assay.Results: after 8 weeks immunization, antibody IgA in E2 vaccine group andE2 +IL-12 vaccine group was statistically increased as compared with that of control group, and the difference was statistically significant (P<0.01). IFN-γand IL-4 levels of mice spleen lymphocyte culture supernatant inE2 group and E2 + IL-12 were significantly higher than other control groups (P<0.01). the spleen lymphopoiesis activeness of E2 + IL-12 group was significant enhanced as compared with the E2 group, there were statistically significant differences (P<0.01) . Conclusions: E2 DNA vaccine combined with IL-12 immune mice can effectively induce cell-mediated immunity and humoral immune responses, their capabilities of inducing immune response might be more stronger than that of single one.     

Amyloid β3-10 DNA vaccination suggests a potential new treatment for Alzheimer’s disease in BALB/c mice

Background  Amyloid β_1-42 (Aβ₄₂) peptide vaccination has been proved to be effective in reducing amyloid burden in brain and improving cognitive function in Alzheimer’s disease (AD) mouse models. But the phase II trial of Aβ₄₂ peptide vaccine was halted because of T cell-mediated meningoencephalitis. In this study, a DNA vaccine, p(Aβ_3-10)₁₀-CpG, was constructed to test whether it would induce predominant T_H2 immune response upon immunization of BALB/c mice.

Methods  BALB/c mice were vaccinated intramuscularly with p(Aβ_3-10)₁₀-CpG plasmids. Aβ₄₂ peptide, pcDNA3.1(+) empty vector and PBS were injected to the control groups. Expression of interesting gene in injected muscle was identified by immunohistochemistry. Anti-Aβ antibody titers, isotype profiles as well as cytokines in ex vivo splenocytes culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA).

Results  P(Aβ_3-10)₁₀-CpG plasmid was expressed in muscle after injection detected by immunohistochemistry. The p(Aβ_3-10)₁₀-CpG vaccine induced high titers of anti-Aβ antibodies in BALB/c mice. And isotype of the antibodies was mainly IgG1, the IgG1/IgG2a ratio for the p(Aβ_3-10)₁₀-CpG group was approximately 5 times greater than that for the Aβ₄₂ peptide group. Ex vivo cultured splenocytes isolated from mice immunized with p(Aβ_3-10)₁₀-CpG exhibited high interleukin-4 response and low interleukin-γ (IFN-γ) response.

Conclusions  Immunization with p(Aβ_3-10)₁₀-CpG vaccine primarily induces a T_H2 type of response, thus reduces the probability of inflammation. This p(Aβ_3-10)₁₀-CpG vaccine possesses the basic factors required for a safe and effective AD vaccine.

     

Immunity induced by DNA vaccine of plasmid encoding the rhoptry protein 1 gene combined with the genetic adjuvant of pcIFN-γ against Toxoplasma gondii in mice

Objective To construct the eukaryotic expression recombinant plasmid, pcIFN-γ, as a gene tic adjuvant and observe the immune responses elicited by pcDNA3-rhoptry protei n 1 （pc-ROP1） combined with pcIFN-γ against Toxoplasma gondii (T.go ndii) infection in mice.Methods A fragment of the IFN-γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion.pcIFN and pcROP1 DNA wa s injected into the left leg muscle of mice at a dosage of 100 μg, and a boost er vaccination was given at the same dosage after two weeks.Control groups wer e injected with pcDNA3 blank plasmid or normal saline.At 30, 50 and 70 days af ter booster injection, kinetic tests were carried out: MTT assay for the prolife ration response of T lymphocyte cells and the activity of NK cells, sandwich ABC -ELISA for the determination of IFN-γ, IL-2 and IL-10; a serum enzymetic aa ssay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera.Results The recombinant plasmid, pcIFN-γ was constructed.The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN- γ, IL-2 and NO in mice injected with pcROP1 and pcIFN-γ were higher than in those injected with pcROP1 alone.There was no difference in IgG antibody level s between the two groups. Conclusion The genetic adjuvant, pcIFN-γ, could enhance the cellular immune response indu ced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection.     

人乳头瘤病毒16型E6E7基因协同共激活分子B7-1基因免疫诱导的特异性免疫反应

目的利用突变修饰后的中国山东地方株人乳头瘤病毒16型(humanpapillomavirustype16,HPV16)E6E7融合基因(fmE6E7),研究治疗HPV16感染相关疾病的DNA疫苗,并进一步探索利用共激活分子B7-1基因,研究更加活化细胞免疫的加强疫苗。方法将用PCR法扩增获得fmE6E7基因插入真核表达质粒pVR1012中获得pVR1012-fmE6E7,瞬时转染Cos-7细胞,免疫荧光组织化学法检测证实其表达后,在C57BL/6小鼠肌肉内进行pVR1012-fmE6E7单独免疫,或与小鼠共激活分子B7-1基因真核表达质粒(pcDNA3.1-B7-1)联合免疫。51Cr释放法分析免疫小鼠的细胞毒性T淋巴细胞(cytotoxicTlymphocytes,CTL)活性,间接ELISA法检测小鼠血清中E7特异性抗体。用5×105个C3细胞皮下接种C57BL/6小鼠,分析小鼠体内诱发的特异性抗瘤免疫水平。结果修饰后的E6E7基因免疫可诱导机体产生特异的抗体反应和CTL反应,小鼠B7-1基因与fmE6E7联合免疫可显著提高特异性CTL活性,并可保护33%(2/6)的小鼠免受C3肿瘤细胞的攻击,而单独fmE6E7基因免疫则不能抑制C3瘤细胞的生长,联合B7-1基因免疫对诱发的抗体水平无加强作用。结论中国山东地方株E6E7融合基因可用于DNA疫苗的构建,B7-1基因协同免疫可提高疫苗的细胞免疫水平,利用B7-1基因作为HPV16DNA疫苗的协同因子具有重要价值。     

Immune response to plasmid DNA encoding HPV16-L1 protein

Objective To test the immunogenicity of recombinant plasmid DNA containing human papillomavirus type 16-L1 (HPV16-L1) coding sequence of mice.Methods The HPV16-L1 encoding sequence was generated by polymerase chain reaction (PCR), and inserted into TA cloning vector PCR Ⅱ, then cloned in the eukaryotic expression vector pcDNA3.1 with CMV promoter. The recombinant plasmid DNA pcDNA-L1 was transferred into Cos-7 cells and used to immunize BALB/c mice via muscular injection. The expression of HPV16-L1 in transferred cells was identified by immunospot and immunocytochemistry, which tested specific anti-HPV16-L1 antibody in the serum of immunized mice. Results Using the immunospot technique, we found L1 protein expression in pcDNA-L1 transferred cells. The immunocytochemistry studies demonstrated that the L1 protein was located in nuclei. In immunized mice, specific anti-HPV16-L1 antibodies could be detected by immunospot and immunocytochemistry 28 days after the first immunization and last at least 41 days. Conclusions We constructed HPV16-L1 eukaryotic expressing plasmid whose DNA could induce immuno-humoral response in mice. This observation will be helpful in designing HPV16 prophylactic vaccine.     

CEA串连表位-HSP70融合基因疫苗诱导小鼠的表位特异性免疫应答

目的：观察癌胚抗原(CEA)串联表位-HSP70融合基因疫苗诱导的免疫应答，为开发新型肿瘤特异性基因疫苗奠定基础。方法：在已经构建含有变异热休克蛋白(HSP)序列的基础上，插入重组CEA串联表位的编码片段获得CEA串联表位-HSP融合基因疫苗。3次肌肉注射免疫Balb/c小鼠，设立注射生理盐水的阴性对照组、注射氢氧化铝佐剂混悬CEA串联表位的阳性对照组及注射pCITriCEA_625-667-mtHSP70的实验组。FCM分析脾脏T细胞亚群；体外培养脾细胞，ELISA检测培养上清中干扰素γ(IFNγ)的相对含量；同时测定小鼠血清CEA特异性抗体的滴度。结果：阴性对照组脾细胞中CD3⁺和CD4⁺T细胞分别为55.1%±6.1%和30.2%±4.1%；实验组脾细胞中CD3⁺和CD4⁺T细胞分别为78.7%±9.2%和48.9%±4.7%，两组比较差异有显著性(P<0.01)。其体外特异性抗原肽诱导的IFNγ分泌处于本底水平，可视为生理性参数，体外非特异性和特异性的IFNγ分泌分别增加3和6倍(P<0.01)。血清中CEA表位特异性抗体滴度阴性对照组为0，阳性对照组＜1∶500,而融合基因疫苗免疫组达到1∶4 000。结论：CEA串联表位-HSP融合基因疫苗在体内诱导以激发辅助性T细胞为特征的免疫应答。     

GM-CSF表达质粒对pcDNA3/MDC-VP1 DNA疫苗的增强作用

目的:观察粒-巨噬细胞集落刺激因子(GM-CSF)对pcDNA3/MDC-VP1DNA疫苗免疫作用的影响,为柯萨奇病毒B组3型(CVB3)疫苗的研制提供理论和实验依据。方法:4～6周龄雄性BALB/c小鼠随机分成pcDNA3组、pcDNA3/MDC-VP1组、pcDNA3/mGM-CSF组、pcDNA3/MDC-VP1与pcDNA3/mGM-CSF混合注射组,每组10只。每3周接种1次,共3次。每次接种的第20天眼眶采血,用微量中和试验(固定病毒-稀释血清法)检测血清中和抗体效价。第3次免疫后3周,每组取3只小鼠脾脏制备淋巴细胞悬液,检测淋巴细胞增殖活性与特异性细胞毒性T淋巴细胞(CTL)杀伤活性。结果:pcDNA3/MDC-VP1+pcDNA3/mGM-CSF组的血清中和抗体滴度明显提高,小鼠脾脏淋巴细胞增殖活性和特异性CTL杀伤活性均有增强。结论:GM-CSF可以增强pcDNA3/MDC-VP1DNA疫苗的特异性免疫应答。     

单纯疱疹病毒1型截短糖蛋白B基因疫苗的构建及其诱导小鼠细胞免疫应答

目的:构建、制备单纯疱疹病毒1型截短糖蛋白B DNA疫苗，检测其诱导机体产生的细胞免疫应答效果。方法:利用PCR技术从HSV-1 SM44毒株基因组中扩增出编码HSV-1 gB14～507氨基酸序列的一段基因。定向插入真核表达质粒pcDNA₃载体中，构建出重组真核表达质粒pcDNA₃-gBt，并对其进行酶切分析、PCR鉴定及测序鉴定。于BALB/c鼠注射免疫3次，抗体分析CD4⁺、CD8⁺T细胞亚群的变化，羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)/碘化丙碇(PI)双标记的流式细胞计数法检测CTL活性。结果:PCR及测序鉴定结果显示，插入的克隆基因与GenBank中HSV-1F株gB基因序列一致，证实了HSV-1 gBt核酸疫苗的构建；pcDNA₃-gBt免疫组BALB/c鼠的CD4⁺T细胞数较空质粒（pcDNA₃）对照组和生理盐水对照组增加，CTL活性也较对照组明显增强，但是pcDNA₃-gBt免疫组BALB/c鼠的CD8⁺T细胞、CD4⁺/ CD8⁺T细胞比值与对照组比较差异均无显著性（P＞0.05）。结论:HSV-1 gBt核酸疫苗诱导较强的细胞免疫应答。